In eukaryotes, protein synthesis (translation) occurs in a complex process in which messenger RNA (mRNA) carrying amino acid sequence information encoded in its nucleotide sequence interacts with ribosomes and a variety of cofactors and enzymes. Among the critical interactions are those which occur in the initial steps of mRNA recognition during initiation of translation
Synthesis of mRNA occurs in the nucleus of the eukaryotic cell. Translation occurs in the cytoplasm. RNA sythesized in the nucleus is subject to modifications, generally termed processing reactions. These include capping, intron splicing and polyadenylation. Of importance herein is the processing step known as capping. Capping is the addition, at the 5xe2x80x2 end of mRNA, of 7-methyl guanine, (m7G) joined by an unusual 5xe2x80x2xe2x80x945xe2x80x2 diphosphate bridge to the 5xe2x80x2 terminal ribonucleotide of mRNA. The capping reaction occurs naturally in the cell nucleus during mRNA synthesis. Capping can also be carried out in vitro in an enzyme-catalyzed reaction. Commercially available kits can be obtained, for example, from Life Technologies, Inc., Gaithersburg, Md.
The initiation of translation in the cytoplasm requires specific binding of proteins termed initiation factors. An important initiation factor in mammalian cells is the eukaryotic Initiation Factorxe2x80x944E (eIF-4E) which binds to capped RNA (m7G-RNA). Translation is regulated in vivo by factors and conditions which affect the binding of eIF-4E to m7G-RNA, including proteins that bind to eIF-4E (4E binding proteins). For example, at least one 4E binding protein designated 4E-BP-1 acts to prevent the binding of eIF-4E to m7G-RNA. 4E-BP-1, also known as PHAS-1, can undergo phosphorylation which is induced by insulin or other growth factors. The insulin-induced phosphorylation of 4E-BP-1 releases the bound eIF-4E which is now available to bind m7G-RNA. This process may account for the rapid stimulation of protein synthesis in muscle tissue induced by insulin. Another eIF-4E binding protein is p220, also known as eIF-4F, a protein that binds with eIF-4E as part of a functional complex which interacts with mRNA to positively regulate translation.
The sequence of DNA encoding human eIF-4E has been determined [Reychlik, W. et al. (1987) Proc. Natl. Acad. USA 84:945-949]. Yeast eIF-4E and a fusion protein of mouse eIF-4E have been expressed in E. coli [Edery, I., et al. (1988) Gene 74:517-525; Edery, I., et al. (1995) Mol. Cell. Biol. 15:3363-3371]. Haas, D. W. et al. (1991) Arch. Biochem. Biophys. 284:84-89 reported purification of native eIF-4E from erythrocytes. Stern, B. D. et al. (1993) reported isolation of recombinant eIF-4E using denaturing concentrations of urea. However, expression and purification of recombinant human eIF from the soluble fraction without a denaturation step was not described before.
Transfection using RNA has not been widely reported. The primary difficulty is the susceptibility of RNA to RNAses and the lack of RNA restriction enzymes and ligases that has prevented in vitro recombination of RNA segments. Nevertheless, transfection with RNA has several advantages over transfection with DNA. Transfection by RNA does not normally lead to genetic alteration of host cells. Instead, a transient expression of the protein encoded by the transfecting RNA is observed. There are circumstances where such transient expression is preferable. For example, RNA transfected cells can transiently express an antigen in an individual to be immunized. Garrity, R. R., et al. (1996) (Abstr. 1996 Meeting on Molecular Approaches to the Control of Infectious Diseases, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., Sep. 9-13, 1996) reported that antibodies to gp 120 and gp 160 of HIV-1 were detectable in guinea pigs that had been injected intramuscularly with naked m7G-RNA encoding the respective antigens. Titres were low and the antibodies did not neutralize homologous virus. Since DNA transfection leads to chromosomal integration of extraneous DNA and long-lived expression of its encoded protein, unpredictable and deleterious effects may occur in the host. Transient expression resulting from RNA transfection can avoid these concerns. The problems to be overcome with RNA transfections include extremely low transfection efficiency and short intracellular lifetime of transfected RNA.
eIF-4E has recently been shown to play a direct role in maintaining the phenotype of breast cancer cells. The levels of eIF-4E in biopsies of breast cancer and breast cancer cell lines are increased (3-30 fold; mean of 10.5xc2x10.9) as compared to benign fibroadenomas of breast tissue and control cells [Kerekatte, V. et al. (1995) Int J Cancer 64(1):27-31; Anthony, B. et al. (1996) Int J Cancer 65:858-863]. Immunohistochemical studies showed that the cells expressing high levels of eIF-4E are indeed cancer cells and not stromal cells. In addition, evidence indicates that high levels of expression of eIF-4E correlate with a poor clinical outcome in breast cancer [Li, B. D. L. et al. (1997) Cancer 79(12):2385-2390]. A direct role for eIF-4E in breast cancer is evidenced by studies demonstrating that mammary carcinoma cells (MDA-435) exhibiting a 50% decrease in eIF-4E expression, due to stable transformation with an antisense construct, have a markedly reduced ability to produce tumors in nude mice. In addition, the down-regulation of eIF-4E expression in these cells results in relatively avascular tumors compared to control cells [Nathan, et al. 1997].
The cocrystal structure of mouse eIF-4E bound to m7GDP [Marcotrigiano J. et al. (1997) Cell 89:951-961] and the solution structure of yeast eIF-4E bound to m7GDP as determined by NMR spectroscopy [Matsuo H. et al. (1997) Nature Struct Biol.4:717-724] have been described. Both studies describe a cap-binding slot for eIF-4E in which the m7G moiety is sandwiched between the side chains of two tryptophans, Trp-56/Trp-102 in mouse and Trp-58/Trp-104 in yeast eIF-4E. A third tryptophan, Trp-166 (both mouse and yeast), as well as Glu-103 in mouse and Glu-105 in yeast, form hydrogen bonds with m7G. The cocrystal structure demonstrated additional interactions involving residues Arg-157, Arg-112, and Lys-162 which make direct or water-mediated contacts with the phosphate groups of m7GDP. The NMR solution structure of yeast eIF-4E showed that Arg-157, Lys-158 and Glu-159 are close to the phosphate tails of m7GDP and M7GTP.
The invention provides purified recombinant human eIF-4E, as well as a method of purification from transgenic cells expressing eIF-4E. Purified wild-type human eIF-4E binds in vitro to m7G-RNA with a binding constant of 10.1xc2x10.3xc3x97105Mxe2x88x921. Binding is 1:1 on a molar basis, forming a binary complex designated eIF-4E-m7G-RNA. A sequence of amino acids involved in binding human eIF-4E to m7G-RNA has been identified. Amino acid substitutions within the eIF-4E amino acid sequence have been made, some of which can result in 1-2 orders of magnitude tighter binding, others of which result in reduced binding. The invention therefore also provides modified human eIF-4E. The term xe2x80x9cvariant human eIF-4Exe2x80x9d as used herein embraces amino acid substitutions, deletions and insertions and combinations thereof affecting the binding affinity of the variant eIF-4E to m7G-RNA without destroying the protein""s capacity to function as an initiation factor.
The wild-type and variant human eIF-4E bound to m7G-RNA improves stability of m7G-RNA, which enhances the transformation efficiency. Furthermore the presence of bound eIF-4E ensures immediate and efficient translation in the transfected host cell, which can be observed as enhanced expression of the protein encoded by the transfecting RNA.
The invention therefore provides a method for making eIF-4E-m7G-RNA and a method for transfecting eukaryotic cells by contacting the cells with eIF-4E-m7G-RNA. The method can be used with variant human eIF-4E or wild-type human eIF-4E. RNA transfected cells transiently express the protein encoded by the RNA sequence.
The invention further provides a method for isolating 4E binding proteins (4E-BP). Immobilized eIF-4E acts as an affinity ligand for the various proteins that bind to it and regulate translation. The 4E-BP proteins can thereby be isolated and characterized, in order to better understand their role in controlling translation.
The invention further provides amino acid sequence variants of human eIF-4E having either reduced or enhanced binding affinity for capped mRNA (m7G-RNA). Variants having reduced binding affinity are useful in treatment of breast cancer. For example, DNA encoding a reduced-binding variant, introduced into breast cancer cells by a gene-therapy technique can act as a dominant negative mutant, counteracting the overexpression of eIF-4E required to maintain the tumor phenotype of such cells. Variants having enhanced binding affinity have increased stability in vitro and in vivo, for improved transient expression of a selected gene in RNA transfection. Similar uses of natural or varied sequence eIF-4E for temporally-limited gene regulation can be recognized by those skilled in the art, including, for example, to control the differentiation of stem cells. As a further utility, the ability of host cells to express large proteins transgenically can be enhanced by transfection using natural or variant eIF-4E.